Generation of antigen-specific memory CD4 T cells by heterologous immunization enhances the magnitude of the germinal center response upon influenza infection

Current influenza vaccine strategies have yet to overcome significant obstacles, including rapid antigenic drift of seasonal influenza viruses, in generating efficacious long-term humoral immunity. Due to the necessity of germinal center formation in generating long-lived high affinity antibodies, the germinal center has increasingly become a target for the development of novel or improvement of less-efficacious vaccines. However, there remains a major gap in current influenza research to effectively target T follicular helper cells during vaccination to alter the germinal center reaction. In this study, we used a heterologous infection or immunization priming strategy to seed an antigen-specific memory CD4+ T cell pool prior to influenza infection in mice to evaluate the effect of recalled memory T follicular helper cells in increased help to influenza-specific primary B cells and enhanced generation of neutralizing antibodies. We found that heterologous priming with intranasal infection with acute lymphocytic choriomeningitis virus (LCMV) or intramuscular immunization with adjuvanted recombinant LCMV glycoprotein induced increased antigen-specific effector CD4+ T and B cellular responses following infection with a recombinant influenza strain that expresses LCMV glycoprotein. Heterologously primed mice had increased expansion of secondary Th1 and Tfh cell subsets, including increased CD4+ TRM cells in the lung. However, the early enhancement of the germinal center cellular response following influenza infection did not impact influenza-specific antibody generation or B cell repertoires compared to primary influenza infection. Overall, our study suggests that while heterologous infection or immunization priming of CD4+ T cells is able to enhance the early germinal center reaction, further studies to understand how to target the germinal center and CD4+ T cells specifically to increase long-lived antiviral humoral immunity are needed.


Introduction
Despite the availability of a vaccine, seasonal influenza infection continues to be a significant burden on the healthcare system in the United States, causing acute respiratory illness and leading to exacerbation of severe health conditions, hospitalization, and mortality [1][2][3].While current seasonal influenza vaccines prevent millions of influenza-related illness cases each year [2], they fail to induce long-term strain-specific immunity due to waning neutralizing antibody titers within a year post-vaccination [4][5][6][7][8][9].In addition, current influenza vaccines do not induce sufficient breadth of cross-reactive neutralizing antibodies to protect against novel variants that arise due to the rapid antigenic drift of the immunodominant globular head of the hemagglutinin (HA) surface glycoprotein [10][11][12][13][14]. Currently, one of the major priorities in the development and improvement of influenza vaccines is to generate broad cross-reactive neutralizing antibody responses [15][16][17][18], though mechanisms driving the generation of these antibodies after viral infection or vaccination are not well understood.
Formation of the germinal center (GC) during an immune response is necessary for generating long-lived humoral immunity, making it an important target for development of novel vaccines or the improvement of lower efficacy vaccines, including the seasonal influenza vaccine.The GC is where the critical processes for generating long-lived humoral immunity occur, including somatic hypermutation, selection for high affinity antibodies, class switch recombination, and generation of memory B cells and long-lived plasma cells [19,20].T follicular helper (Tfh) cells are the primary CD4+ T cell subset that helps B cells promote the GC reaction [19][20][21][22][23][24], and are required to produce long-lived humoral immune responses.Tfh cells are mainly distinguished by expression of the B cell follicle homing receptor CXCR5 [25-
Previous studies have shown that increased circulating memory Tfh cells in HIV-infected patients and highly functional GC Tfh cells in HIV-immunized rhesus macaques correlate with enhanced production of broadly neutralizing antibodies [43][44][45].In influenza-related studies, adjuvanted inactivated influenza vaccine was shown to increase GC responses and enhance cross-reactivity and long-term detectability of HA-specific antibodies [46].In addition, vaccination with influenza HA-ferritin nanoparticles showed a positive correlation between increased ferritin-specific CD4+ GC Tfh cells and increased HA-specific GC B cells and antibody secreting cells [47].Overall, these studies suggest that current influenza vaccines are unable to induce universal broad cross-reactive anti-influenza neutralizing antibodies, possibly in part due to poor accessibility or epitope masking of conserved epitopes by pre-existing antibodies [11,14,[48][49][50].Given that Tfh cells have been shown to be the limiting cell subset in the GC reaction [51][52][53], as well as Tfh cell magnitude correlating with formation of broadly neutralizing antibodies [43][44][45][46], we proposed that by directly manipulating Tfh cell magnitude in mice via heterologous infection or immunization priming of CD4+ T cells we would enhance the germinal center reaction and its products compared to primary influenza infection alone.
In this study, we generated antigen-specific CD4+ Tfh memory cells using heterologous priming with either intranasal (i.n.) infection with acute lymphocytic choriomeningitis virus (LCMV) or intramuscular (i.m.) immunization with adjuvanted recombinant glycoprotein from LCMV (rGP) prior to intranasal infection with a recombinant mouse-adapted PR8 strain engineered to carry the CD4-immunodominant LCMVgp61-80 epitope (PR8-HA-GP 61-80 ).We then assessed the GC response and antibodies following influenza challenge.We found that heterologous influenza rechallenge resulted in significant increases in the numbers of polyclonal effector antigen-specific CXCR5-Th1 cells in both rGP-and LCMV-primed mice, as well as CXCR5+BCL6+ GC Tfh cells in LCMV-primed mice compared to primary influenza infection.In addition, we analyzed lung-resident CD4+ T cells following heterologous influenza rechallenge and found a significant bias in resident Th1-like cells in LCMV-primed mice and in resident Tfh-like cells in rGP-primed mice, as well as a significant increase in the longterm CD4+ T resident memory pool compared to primary influenza infection.While heterologous infection or immunization priming of CD4+ T cells was able to enhance the early GC cellular response following influenza challenge, we did not see corresponding increases in generating long-term HA-specific antibodies or antibody-secreting cells.Along with previous studies showing the importance of CD4+ Tfh cells in GC and formation of high affinity humoral immunity, our findings suggest that targeting the expansion of memory CD4+ T cells to enhance the primary GC B cell response and tissue-resident memory population is possible and could be a promising avenue to the expansion of memory generation in next generation influenza vaccines.

Ethics statement
All animal experiments were conducted in accordance with protocols that were approved by the University of Utah Institutional Animal Care and Use Committee.

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Pre-made memory CD4+ T cells promote enhanced GC responses to flu

Viral infections and protein immunizations
C57BL/6J mice (Jackson Laboratory, Bell Harbor, ME) were infected with either 30 μl of 500 TCID 50 mouse-adapted PR8-HA-GP 61-80 or 2x10 5 PFU of LCMV Armstrong by intranasal inoculation or immunized by intramuscular (quadriceps) injection with 2 μg LCMV recombinant glycoprotein (rGP) with addition of Addavax (InvivoGen) adjuvant at a 1:1 ratio.PR8-HA-GP 61-80 recombinant virus strain is the H1N1 PR8 strain with the CD4-immunodominant LCMVgp61-80 epitope inserted into the HA region and was kindly provided by Dr. Florian Krammer (Icahn School of Medicine at Mount Sinai).293A cells that express recombinant glycoprotein (from LCMV) were kindly provided by Dr. Carl Davis (Emory University), and recombinant glycoprotein was purified from supernatants as described previously [54].For B cell reactivation experiments, mice were immunized by intraperitoneal injection with 10 μg recombinant HA (H1 subtype) protein from PR8 (H1N1) virus without adjuvant.For intranasal infections, mice were anesthetized with concurrent administration of aerosolized isoflurane and oxygen using a COMPAC 5 Anesthesia Center (VetEquip).Prior to euthanasia, mice were intravenously injected by retro-orbital injection with 2 μg α-CD45-FITC (30-F11, Tonbo Biosciences) antibody to detect remaining circulating cells in lung samples [55].

Construction of the recombinant influenza virus PR8-HA-GP 61-80
To obtain a recombinant influenza virus containing the LCMV epitope GP 61-80 (GLKGPDIYKGVYQFKSVEFD) inserted in the hemagglutinin (HA) protein, the sequence encoding the GP 61-80 peptide was introduced in the rescue plasmid pDZ-HA, strain A/Puerto Rico/8/1934 (H1N1) (PR8).The epitope was inserted in-frame at the amino acid position 135, that is highly tolerant to small insertions [56].Next, the recombinant virus was rescued by transfecting cells with 8 plasmids containing the sequences of the viral segments, as previously described [57].

Tissue processing
Single-cell suspensions of pooled mediastinal lymph nodes or pooled inguinal and lumbar lymph nodes were prepared using 70-μm cell strainers.Single-cell suspensions of spleens were prepared using 70-μm cell strainers and red blood cells lysed by incubation in Ammonium-Chloride-Potassium (ACK) Lysing Buffer (Life Technologies).Single-cell suspensions of lungs were prepared by digestion with 0.25mg/ml Collagenase IV and 15 μg/ml DNase for 1 hour at 37˚C, then manually homogenized and red blood cells lysed by incubation in ACK Lysing Buffer and then cells were filtered using 70-μm cell strainers.Cell suspensions were resuspended in RPMI 1640 media supplemented with 5% fetal bovine serum (FBS) prior to FACS staining.

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Pre-made memory CD4+ T cells promote enhanced GC responses to flu staining was performed using a three-step protocol described in Johnston et al. (2009) [29] using purified rat anti-mouse CXCR5 primary antibody (BD Biosciences, 2G8) in FACS buffer supplemented with 1% bovine serum albumin (Sigma, #A7284) and 2% normal mouse serum (Sigma, #M5905) (CXCR5 staining buffer), a secondary Biotin-SP-conjugated Affinipure F (Ab') 2 Goat anti-Rat IgG (Jackson ImmunoResearch) in CXCR5 staining buffer and then with a fluorochrome-conjugated streptavidin in FACS buffer.For transcription factor staining, cells were first stained for surface antigens, followed by permeabilization, fixation and staining using the Foxp3 Permeabilization/Fixation kit and protocol (eBiosciences).Intracellular cytokine staining was done by standard techniques following 5-hour stimulation with Gp 61-80 peptide and Brefeldin A (GolgiPlug, BD Biosciences).No peptide controls were treated under the same conditions supplemented with Brefeldin A but without Gp 61-80 peptide.Cells were then stained for surface antigens, followed by permeabilization, fixation and staining using the Cytofix/Cytoperm kit and protocol (BD Biosciences).For influenza HA-specific B cell staining, recombinant HA protein from A/Puerto Rico/8/1934 (H1N1) virus strain (Immune Technology Corp., #IT-003-0010ΔTMp) was biotinylated with 80-fold molar excess of NHS-PEG4-Biotin solution from the EZ-Link NHS-PEG4-Biotin kit (ThermoFisher, #A39259).Excess biotin was removed by buffer exchange of protein into sterile 1X PBS using Zeba Spin Desalting Columns, 7K MWCO (ThermoFisher, #89882).Cells were stained on ice for 30min in FACS buffer with 1:100 dilutions of biotin-conjugated-HA and purified rat antimouse CD16/CD32 (Mouse BD Fc Block, Clone 2.4G2, BD Biosciences), then stained on ice for 30min in FACS buffer with 1:1000 dilution of allophycocyanin (APC)-conjugated streptavidin.Cells were analyzed on LSRFortessa X-20 and LSRFortessa (BD Biosciences) cytometers.FACS data were analyzed using FlowJo v10 software (Tree Star).

Hemagglutination inhibition assay (HAI)
Serum was separated from whole blood by centrifugation at 10,000xg for 30min at 4˚C.HAI to determine neutralizing antibody titers was performed by incubating 25 μL of two-fold serially diluted serum with 25 μL of 4 agglutinating doses (4AD) of WT PR8 (H1N1) virus strain for 30min at room temperature (RT) prior to addition of 50μL of 1% chicken red blood cells (cRBCs) (Lampire Biological Laboratories) in 1X PBS.Plates were gently agitated to mix and then incubated for 30min at RT. HAI titers were determined as the reciprocal dilution of the final well which contained non-agglutinated cRBCs.Naïve mouse serum was used as a negative control.Mice with titers of <1:10 were not included in final analyses.

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Pre-made memory CD4+ T cells promote enhanced GC responses to flu hydrochloric acid and plates were scanned at 490nm using a Biotek Synergy H1 microplate reader.Naïve mouse serum was used as negative controls.OD readings were averaged between technical duplicates for all samples.Titer cutoff value was determined using the OD values of negative controls as described in Frey et al. (1998) [58] using a 95% confidence level.Relative endpoint titers were calculated by nonlinear regression interpolation of a standard curve (Sigmoidal, 4PL, X is concentration) of individual samples using GraphPad Prism version 9.4.1 for macOS and calculating the titer at which each curve crosses the background cutoff value.

Enzyme-linked immunosorbent spot assay (ELISpot)
Bone marrow was collected from femur and tibia bones and red blood cells lysed by incubation in ACK Lysing Buffer.B cell enrichment of bone marrow cells was performed using the Pan B Cell Isolation Kit (Miltenyi Biotec, #130-095-813).MultiScreen-IP Filter Plates (Sigma, #MAIPS4510) were pre-wet with 15 μL 35% ethanol for 30 sec and washed with 1X PBS.Plates were coated with 2 μg/mL of recombinant HA protein from A/Puerto Rico/8/1934 (H1N1) virus strain (Immune Technology Corp.) overnight at 4˚C.Plates were washed with 1X PBS and then blocked for 2hr at RT with RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% Penicillin-Streptomycin, 2 mM L-glutamine (complete culture medium).Plates were washed with 1X PBS and then enriched B cells in complete culture medium were added to plates at two-fold serial dilutions and in technical duplicates for each sample at maximum 4x10 6 cells/well and incubated overnight at 37˚C at 5% CO 2 .Plates were washed with 1X PBS, then washed with 1X PBS with 0.05% Tween 20, then incubated with Goat anti-Mouse IgG (H +L) Cross-Adsorbed Secondary Antibody conjugated to HRP (ThermoFisher, #G-21040) at a 1:350 dilution in 1X PBS with 0.05% Tween 20 and 1% fetal bovine serum overnight at 37˚C at 5% CO 2 .Plates were washed with 1X PBS with 0.05% Tween 20, then washed with 1X PBS, and plates were developed using the AEC Staining Kit (Sigma, #AEC101-1KT).After spot development, plates were washed with water and allowed to dry before counting.

Histology and immunofluorescence
To analyze influenza infected lungs by histology, mice were humanely euthanized by CO 2 and perfused with PBS (Thermo Fisher Scientific) through the left ventricle of the heart.Lungs were then inflated with 4% paraformaldehyde, removed, and placed in 4% paraformaldehyde at 4˚C overnight.The next day, the left lung was carefully dissected and stored in 4% paraformaldehyde at 4˚C for 24 hours, while the right lung was carefully dissected and stored in 70% ethanol.After 24hrs, the right lung was sent to the University of Utah Biorepository and Molecular Pathology (BMP) Shared Resource for paraffin embedding, sectioning, and staining with hematoxylin and eosin.The left lung was placed into a 30% sucrose solution in PBS at 4˚C for 2 days before being placed in a 10% sucrose solution for 4 hours.Next, lungs were washed with PBS at RT with agitation for 1 hour before being embedded in OCT (Tissue-Tek) and flash frozen.Twenty-micron cryosections were sliced from each sample and stored at 80˚C until staining.Before staining, sections were fixed using a mixture or ethanol and acetone, rehydrated with PBS, and blocked using 5% rat and 5%rabbit serum in PBS.After blocking for an hour at 4˚C, sections were stained with the following antibodies from Invitrogen overnight at 4˚C: CD4-FITC (RM4-5), CD31-PE (390), B220-AF561(RA3-6B2). Slides were then washed 5x with PBS and agitation before mounting with Fluoromount-G (Thermo Fisher Scientific).Samples were imaged using a Leica SP8 DIVE.Tile scans were obtained at 1048x1048 resolution and z-stacks are represented by maximum projection.

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Pre-made memory CD4+ T cells promote enhanced GC responses to flu

B cell repertoire sequencing
Single-cell suspensions of splenocytes from individual mice were prepared and B cell enrichment was performed using the Pan B Cell Isolation Kit (Miltenyi Biotec, #130-095-813).Plasmablast cell sorting was performed using a FACSAria (BD Biosciences).Genomic DNA was isolated from sorted plasmablasts using QIAamp DNA Mini Kit (Qiagen), and amplification and sequencing of the Igh locus were performed using the immunoSEQ platform (Adaptive Biotechnologies).Data analyses were conducted in the immunoSEQ Analyzer (Adaptive Biotechnologies) and R [59], RStudio [60], and the Immunarch package [61].Data was exported from RStudio using the writexl package [62].

Statistical analysis
All experiments were analyzed using GraphPad Prism version 9.

Generation of antigen-specific memory Tfh cells in rGP-immunized and LCMV-infected mice prior to influenza challenge
Our goal was to generate antigen-specific memory CD4+ T cells by heterologous priming with protein immunization or viral infection that could provide help during a primary influenza response.We first evaluated the kinetics and differentiation of polyclonal antigen-specific CD4+ T cells following adjuvanted protein immunization or acute viral infection.C57BL/6J mice were primed by i.m. immunization with 2 μg recombinant LCMV glycoprotein (rGP) in AddaVax adjuvant (GP(1˚) group) or by i.n.inoculation with 2x10 5  When we analyzed for IFNγ-expressing memory CD4+ T cells in spleen following gp61-80 peptide restimulation and normalized to IFNγ background expression in naïve CD4+ T cells, there was a significantly higher frequency and number of IFNγ-expressing cells in the LCMV (1˚) group (S1E-S1F Fig) .The lack of IFNγ-expressing cells in the GP(1˚) group was expected, as we previously reported that adjuvanted rGP immunization induces expansion of a CXCR5-

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Pre-made memory CD4+ T cells promote enhanced GC responses to flu IFNγ-nonpolarized T helper cell population in lieu of highly IFNγ-expressing Th1 cells as seen following LCMV infection [63].Together, these data show that both adjuvanted rGP immunization and LCMV infection induced I-A b :gp66-77 tetramer+ memory CD4+ Tfh and non-Tfh cells.
In addition, we wanted to confirm that our heterologous priming did not induce antibody production to HA protein.We analyzed the serum from GP(1˚), LCMV(1˚), and naïve mice, along with mice infected intranasally with PR8-HA-GP 61-80 recombinant influenza virus at 39-42 dpi for antibodies generated against PR8-HA protein.Antibodies were found that bound to PR8-HA-GP 61-80 recombinant protein in the GP(1˚) and the PR8-HA-GP 61-80 primary influenza response (PR8(1˚)) group, but not the naïve or LCMV(1˚) groups (S1G Fig) .Importantly, antibodies that bound to PR8-HA recombinant protein were only found in the PR8(1˚) serum, suggesting that adjuvanted rGP immunization and LCMV infection did not induce primary B cell responses specific to PR8-HA (S1G Fig).

Previously generated antigen-specific memory CD4+ T cells induced increased effector Th1 and GC Tfh cells upon influenza infection
We next evaluated if using heterologous priming with adjuvanted rGP immunization or i.n.LCMV infection to generate antigen-specific memory CD4+ T cells would enhance the early effector germinal center response to influenza infection.Using the heterologous priming strategies detailed in Fig 1A, 42 days after priming infection or immunization we infected i.n. with 500 TCID 50 PR8-HA-GP 61-80 recombinant influenza virus (GP(1˚)PR8(2˚) and LCMV(1˚) For control groups, we used age-and sex-matched naïve mice infected i.n. with PR8-HA-GP 61-80 to evaluate the primary influenza response (PR8(1˚) group) and a separate group was homologously primed with PR8-HA-GP 61-80 i.n.infection (PR8(1˚) PR8(2˚) group) to evaluate recalled cellular and antibody responses (Fig 2A).
8 days after influenza infection, both GP(1˚)PR8(2˚) and LCMV(1˚)PR8(2˚) groups had significantly higher frequencies and numbers of effector tetramer+ CD4+ T cells in medLN than the PR8(1˚) and PR8(1˚)PR8(2˚) groups, with LCMV-primed mice having the highest overall (Fig 2B -2C); while total lymphocytes were mostly comparable across the four groups (S3A Fig) .Heterologous infection or immunization priming prior to influenza infection induced increased frequencies and numbers of effector tetramer+ CXCR5-TBET+ Th1 cells compared to both primary influenza infection alone and influenza-immune mice at 8 dpi (Fig 2D -2E).While PR8(1˚) mice had a significantly higher frequency of CXCR5+PD-1+ Tfh and CXCR5+BCL6+ GC Tfh cells in medLN, the numbers of GC Tfh cells were significantly higher in LCMV-primed mice (Fig 2D and 2F-2G).Although the numbers of BCL6-expressing CXCR5+ GC Tfh cells were highest in LCMV-primed mice, the amount of BCL6 expression in tetramer+ CXCR5+ cells was significantly lower than in both GP-primed mice and after primary influenza infection (Fig 2G -2H).In addition, our data showed distinct populations in medLN of CXCR5+LY6C low Tfh cells and CXCR5-LY6C high Th1 cells, although the Th1 cells in the GP(1˚)PR8(2˚) group were mostly LY6C low (S3B Fig) .Cytokine analysis of CD4+ T cells in medLN following gp61-80 restimulation revealed that LCMV-primed mice had the highest frequency and number of both IFNγ+ and polyfunctional IFNγ+TNFα+IL-2+ expressing cells (S3C-S3E Fig) .The GP(1˚)PR8(2˚) group also had a significantly higher number of IFNγ+ expressing cells than the PR8(1˚) and PR8(1˚)PR8(2˚) groups, further confirming that memory nonpolarized T helper cells can form Th1 cells after secondary heterologous activation (S3D Fig) .We also evaluated differences in tetramer+ Th1 and Tfh cells in the spleen and found that similar to medLN, while total lymphocytes were mostly comparable in number (S3A Fig), Th1 cells (by both TBET and IFNγ expression) in LCMV-primed mice were significantly higher overall (S3F-S3H Fig).However, our data showed no differences in Tfh cells in the spleen by PD-1 or BCL6 expression in the heterologously-primed or primary influenza infection groups (S3I-S3J Fig) .Together, our data show that heterologous priming with rGP immunization or LCMV infection had induced enhanced expansion of CD4+ Tfh and Th1 cells compared to both primary influenza and secondary homologous influenza infection.

Prior generation of memory Tfh cells promoted increased influenzaspecific GC B cells and plasmablasts upon influenza infection
To determine if recalled antigen-specific memory CD4+ T cells in heterologously primed mice could enhance the primary anti-influenza B cell response, we next analyzed GC B cell and plasmablast populations in medLN and spleen.Total CD19+B220+/low B cells in medLN were mostly comparable by frequency and number, with the lowest B cell numbers in the PR8(1˚)

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Pre-made memory CD4+ T cells promote enhanced GC responses to flu However, heterologous infection or immunization priming of CD4 + T cells drove a significant increase in the number of total Fas+GL7+ GC B cells and influenza HA-specific GC B cells (Fig 3A -3D) at 8 dpi.In addition, while the number of total IgD-CD138+ plasmablasts in medLN were similar between the unprimed and heterologouslyprimed groups, there was a significant increase of HA-specific plasmablasts in both rGP-and LCMV-primed mice (Fig 3E-3H).Analysis of the splenic B cell response revealed significant increases in numbers of total GC B cells and plasmablasts, as well as HA-specific GC B cells and plasmablasts in the GP(1˚)PR8(2˚) group compared to the PR8(1˚) group (S4B-S4F Fig), despite no differences in splenic tetramer+ GC Tfh cells (S3I-S3J Fig).
To determine if there was a correlation between the numbers of tetramer+ GC Tfh cells and HA-specific GC B cells in medLN, we performed Spearman correlation analysis and found that in all groups there was a statistically significant positive correlation (Fig 3I).In addition, linear regression analysis of GC Tfh and HA-specific GC B cell numbers showed statistically significant positive associations in the unprimed and heterologously-primed groups (Fig 3I), consistent with previous studies describing the critical interaction between Tfh and B cells in the germinal center [29,51,[64][65][66].We further compared the numbers of HA-specific GC B cells to the number of GC Tfh cells and found that in the GP(1˚)PR8(2˚) and PR8(1˚)PR8(2˚) groups, there were significantly higher numbers of HA-specific GC B cells to every GC Tfh cell (Fig 3J).These data suggest that while the number of GC Tfh cells in the GP(1˚)PR8(2˚) group were similar to the PR8(1˚) group, the GC Tfh cells may be of a higher quality as to sustain support for higher numbers of HA-specific GC B cells, as has been previously suggested [44,47].For the PR8(1˚)PR8(2˚) group, prior studies have shown poor recall of memory CD4 + cells during homologous secondary infection [67], likely causing the higher ratio of HA-specific GC B cells to antigen-specific GC Tfh cells.Together, these data suggest that heterologous priming with adjuvanted rGP immunization or LCMV infection significantly enhanced the early anti-influenza germinal center B cell response following influenza infection compared to primary influenza infection.

Previously generated memory CD4+ T cells by heterologous immunization did not impact anti-influenza antibody titers
To determine if the early increases in tetramer+ CD4+ T cells and influenza-specific B cells in heterologously primed mice were maintained at memory, we analyzed the cellular response longitudinally in medLN and spleen 42 days after influenza challenge as detailed in Fig 2A .Similar to 8 days after influenza challenge, heterologously primed mice maintained significantly higher frequencies and numbers of I-A b :gp66-77 tetramer+ CD4+ T cells in medLN 42 days after influenza challenge compared to the unprimed PR8(1˚) and homologously primed PR8(1˚)PR8(2˚) groups (Figs 4A and S5A).Our data also showed significantly greater frequencies and numbers of tetramer+ CXCR5-TBET+ Th1 cells and significantly higher numbers of antigen-specific polyfunctional cytokine-secreting (IFNγ+TNFα+IL-2+) CD4+ T cells in heterologously primed mice in medLN (Fig 4B -4C).In addition, memory I-A b :gp66-77 tet-ramer+ CD4+ T cells, tetramer+ CXCR5-TBET+ Th1 cells and polyfunctional cytokine secreting CD4+ T cells were detectable in the spleen at significantly higher numbers in heterologously primed mice (S5B-S5E Fig) .As the CD4+ T cell-specific immunodominant LCMVgp61-80 epitope contains a cryptic epitope recognized by CD8+ T cells [68], we analyzed CD8+CD44+ T cells at 8 and 42 days after influenza challenge for IFNγ expression following LCMVgp61-80 peptide restimulation.Our data show that most mice had frequencies of CD44+IFNγ+ cells of CD8+ T cells below background levels as normalized to a no peptide control, and all other mice had less than 1%

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Pre-made memory CD4+ T cells promote enhanced GC responses to flu These data suggest that while non-specific secretion of IFNγ by CD8+ T cells was detected, we do not expect these cells significantly influenced the increases in Th1 cells and IFNγ-secreting CD4+ T cells.
We next analyzed I-A b :gp66-77 tetramer+ CD4+ T cells for CXCR5, PD-1, and BCL6 expression following influenza infection in medLN to determine the kinetics of memory antigen-specific Tfh cells.At 42 days after influenza infection, the PR8(1˚) group had maintained a significantly higher frequency of both PD-1-and BCL6-expressing Tfh cells (Fig 4D -4E) similar to the effector timepoint.While BCL6 expression in memory CXCR5+ Tfh cells is significantly reduced following acute viral clearance compared to effector CXCR5+ GC Tfh cells [69], LCMV(1˚)PR8(2˚) mice still had significantly higher numbers of BCL6-expressing Tfh

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Pre-made memory CD4+ T cells promote enhanced GC responses to flu cells maintained at memory compared to the other three groups (Fig 4E).When we analyzed lymphocytes at 8, 15 and 42 days after influenza infection to evaluate differences in proliferation or contraction kinetics of CXCR5+BCL6+ GC Tfh cells, our data showed no differences in longitudinal kinetics of GC Tfh cells in this experiment and only significantly higher numbers of GC Tfh cells in heterologously primed mice at 8 dpi (Fig 4F).
We then analyzed the B cell populations in medLN at 42 dpi and found only the LCMV(1˚) PR8(2˚) group had significantly higher numbers of HA-specific GC B cells compared to GP(1˚) PR8(2˚) and PR8(1˚)PR8(2˚) mice (Fig 4G).When we analyzed HA-specific GC B cell kinetics at 8, 15, and 42 days after influenza infection, HA-specific GC B cells underwent contraction after peak expansion around 8 dpi in heterologously primed mice (Fig 4H).However, in the PR8 (1˚) group HA-specific GC B cells increased in number after 8 dpi, though numbers were not significantly different from heterologously primed at 15 or 42 dpi in this experiment (Fig 4H).
As heterologous infection or immunization priming significantly increased HA-specific GC B cells and plasmablasts 8 days after influenza infection, we analyzed the sera of influenza infected mice to determine HA-specific neutralizing antibody and IgG antibody titers.We found that LCMV infection had a slight but statistically significant adverse impact on HA-specific IgG antibody titers compared to influenza infection alone (Fig 4I).In addition, despite heterologous infection or immunization priming inducing increased antigen-specific GC Tfh and GC B cells 8 days after influenza infection, all groups had similar HA-specific neutralizing antibody titers at all timepoints (S5G Fig) .To determine if the enhanced early germinal center cellular response in heterologously primed mice corresponded to an increase in HA-specific long-lived plasma cells, we analyzed enriched B cells from bone marrow of infected mice for IgG secretion by ELISpot 42 and 105 days after influenza infection.As with our serology data, we found no differences in HA-specific IgG-secreting B cells from infected mice regardless of priming strategy (S5H-S5I Fig) .Together these data suggest that while heterologous infection or immunization priming of CD4+ T cells did significantly enhance germinal center CD4+ T and B cell responses early after influenza infection, those effects did not significantly impact long-term germinal center-driven humoral responses compared to primary influenza infection.In addition, our data show that both adjuvanted rGP immunization and LCMV infection significantly enhanced the memory antigen-specific Th1 cell pool after influenza infection, despite differences in the CXCR5-non-Tfh cell populations prior to influenza infection.

Prior generation of antigen-specific memory CD4+ T cells enhanced early GC responses and long-term lung-resident Th1 cells upon influenza infection
Recent evidence has indicated an important role for CD4+ T resident memory (T RM ) cells in mediating protection from influenza infection in the lung [70][71][72][73][74]. Lung-resident CD4+ T cell responses result in the formation of T RM with either Th1 or Tfh properties that can coordinate localized immune responses [75,76].Furthermore, lung-specific immune responses are characterized by the induction of localized B cell responses and the formation of long-lived tissueresident memory B cells that primarily home to the bronchoalveolar lymphoid tissue (BALT), and it is likely that localized antibody responses comprise a key line of defense against influenza infection in the lung [77][78][79].To determine the impact of heterologous infection or immunization priming of CD4+ T cells on the establishment and boosting of secondary T RM we assessed CD4+ T cell responses in the lung following influenza infection of primed and unprimed mice as previously described in Fig 2A .We employed intravascular anti-CD45 staining to distinguish lung-infiltrating leukocytes from those in circulation [55], combined with CD69 staining to identify T RM [71].Both primary adjuvanted rGP immunization and

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Pre-made memory CD4+ T cells promote enhanced GC responses to flu LCMV i.n.infection induced small numbers of lung-infiltrating memory I-A b :gp66-77 tetra-mer+ CD4+ T cells detected at 39 dpi (S6A-S6C Fig) that were dramatically boosted in frequency and number at 8 days after influenza infection and were significantly higher compared to the PR8(1˚) and PR8(1˚)PR8(2˚) groups (Fig 5A -5B).The priming strategy utilized also impacted the resulting secondary effector CD4+ T cell subsets.After PR8-HA-GP 61-80 infection, rGP immunization-induced memory CD4+ T cells preferentially gave rise to FR4
Prior to PR8-HA-GP 61-80 infection, we analyzed lung-infiltrating CD4+ T cells for cytokine expression following ex vivo gp61-80 peptide restimulation and found that primary LCMV i.n.infection induced significantly more IFNγ-and TNFα-producing T cells in the lung compared to adjuvanted rGP immunization at 39 dpi (S6D-S6E Fig) similar to our data of CD4+ T cells in the lymph nodes and spleen.When we analyzed effector CD4+ T cells in the lung for cytokine expression 8 days after PR8-HA-GP 61-80 challenge, the LCMV(1˚)PR8(2˚) group had the highest expansion of CD4+ T cells producing IFNγ and TNFα (Fig 5F -5G), despite the presence of similar numbers of total tetramer+ CD4+ T cells to the GP(1˚)PR8(2˚) group (Fig 5B).
We next sought to determine the impact of heterologous infection or immunization priming of CD4+ T cells on the establishment of lung-infiltrating memory CD4+ T cells following influenza infection.As was the case for the secondary effector response in the lung (Fig 5), PR8-HA-GP 61-80 rechallenge of rGP immunization-or LCMV infection-derived memory CD4+ T cells resulted in a large population of tetramer+ secondary memory T cells in the lung at 42 dpi as compared to the PR8(1˚) and PR8(1˚)PR8(2˚) groups (Fig 6A -6B).Most of these cells expressed CD69, a marker of lung CD4+ T RM following influenza infection [71,72], resulting in a 50-100-fold increase in CD4+ T RM following heterologous rechallenge of rGPor LCMV-derived CD4+ memory T cells (Fig 6C -6D).In addition, the memory CD4+ T cells maintained their primary activation-dependent Th1 and Tfh bias, as the LCMV(1˚)PR8(2˚) mice had significantly more LY6C+ Th1-like secondary memory T cells 42 days after influenza infection and the GP(1˚)PR8(2˚) mice had significantly more FR4+ Tfh-like secondary memory T cells (Fig 6E-6G).Overall, our findings showed that heterologous infection or immunization priming of CD4+ T cells induced large numbers of lung T RM following PR8-HA-GP 61- 80 rechallenge, with a Th1-like or Tfh-like subset distribution that was dependent on the primary immunization or infection challenge.

Igh sequencing of reactivated plasmablasts suggests that heterologous priming did not significantly impact the repertoire diversity or shared clones compared to influenza infection alone
To determine if heterologous infection or immunization priming of CD4+ T cells markedly impacted the B cell clonal repertoire selection compared to mice infected with only influenza, we used the priming and influenza challenge experimental setups as previously described in

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Pre-made memory CD4+ T cells promote enhanced GC responses to flu specific memory B cells to analyze secondary plasmablasts derived from the recalled HA-specific B cells.Five days after rHA immunization, IgD-CD19+B220 high/low Fas+CD138+ plasmablasts were sorted from spleens.Gating was determined using naïve control mice that lacked plasmablast formation (Figs 7A and S8A).Genomic DNA was isolated from sorted plasmablasts and Igh amplification and sequencing were performed using the immunoSEQ platform from Adaptive Biotechnologies.
To investigate the overlap of individual mice repertoires, we performed multidimensional scaling (MDS) analysis on CDR3 amino acid sequences using the overlap coefficient of the repOverlap function of the Immunarch [61] package.There was no discernible clustering by priming strategy by MDS analysis (Fig 7B), indicating there was no significant impact on the repertoires of mice primed by the same strategy.We performed Chao1 estimation [80] and found no differences in clonal repertoire diversity richness by priming strategy (Fig 7C).We next analyzed the diversity of the productive rearrangements (rearrangements that produce functional B cell receptors) in individual mice by Simpson clonality measure, which is calculated as the square root of Simpson's Index [81], which suggested that all repertoires skewed more polyclonal than mono-or oligoclonal (Fig 7D).When we compared the number of In addition, we analyzed CDR3 clonotypes for differences in amino acid sequence length and number of somatic hypermutations (SHM) within nucleotide sequences and found no differences when compared by priming strategy (S8C-S8D Fig) .We next performed the Morisita overlap index test [82][83][84][85] on CDR3 amino acid sequences pooled for all 5 mice in each group to evaluate the repertoire overlap by priming strategy.Our data suggest the PR8(1˚) group had the most unique repertoire, while the GP(1˚)PR8(2˚) and LCMV(1˚)PR8(2˚) groups had more similar repertoires to one another (Fig 7E).When we analyzed the amino acid CDR3 sequences of individual mice with the Morisita overlap index test, we found that most of the GP(1˚)PR8(2˚) and LCMV(1˚)PR8(2˚) mice were more similar to each other than mice only infected with influenza (S8E Fig).
To evaluate shared CDR3 sequences and investigate proportional differences in mice by priming strategy, we tracked the largest 10 clonotypes by total proportion and shared in at least 5 of 20 total infected mice (partially shared clonotypes) using the trackClonotypes feature of the Immunarch [61]

Discussion
Current vaccine strategies, including seasonal influenza vaccines, are not specifically designed to engage CD4+ T cells, despite their necessity in germinal center formation and long-lived humoral immunity, as well as their contribution to cellular immunity in infected tissues.In this study we used heterologous priming with adjuvanted rGP intramuscular immunization or LCMV intranasal infection to generate memory CD4+ T cells and investigate the effects of recalled memory CD4+ Tfh cells and established T RM cells on the response to influenza challenge.Our experimental design that utilized intramuscular immunization for priming is of

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Pre-made memory CD4+ T cells promote enhanced GC responses to flu particular importance, as the antigen dose is limited and the dLNs for the i.m. route are remote to the mediastinal LNs, ensuring that the GP 66-77 epitope-specific CD4+ T cell response upon influenza challenge is generated from memory Tfh cells and not residual effector Tfh cells in GCs that may have persisted after acute viral infection.Our findings demonstrated that heterologous infection or immunization priming induced a population of antigen-specific memory CD4+CXCR5+ Tfh cells that were successfully recalled to secondary effector GC Tfh cells and induced an increased magnitude of HA-specific GC B cells compared to primary influenza infection.Furthermore, while LCMV-primed mice had significantly higher GC Tfh cells 8 dpi, our data suggested rGP-immunization priming produced higher quality GC Tfh cells as these mice had a significantly higher ratio of HA-specific GC B cells to GC Tfh cells.Heterologous infection or immunization priming also induced an increase of secondary effector CXCR5-Th1 cells that expressed both TBET and IFNγ, which were maintained at a higher magnitude even at later timepoints.In addition, heterologous infection or immunization priming generated an increased long-lived CD4+ T RM pool and induced increased expansion of recalled antigen-specific CD4+ T cells in the lung after influenza challenge.Interestingly, the skewing of lung-infiltrating CD4+ T cells was dependent on priming activation, as rGP immunizationprimed mice preferentially recalled Tfh-like cells compared to LCMV-primed mice that preferentially recalled Th1-like cells.However, despite the early enhancement of the germinal center cellular response after influenza challenge, heterologous infection or immunization priming of CD4+ T cells did not enhance HA-specific antibody titers.Overall, our findings suggest that heterologous infection or immunization priming of CD4+ T cells can be used to enhance both the early GC response, including the GC Tfh and GC B cell magnitude, and establishment of CD4+ T RM cells that respond to influenza challenge.
Tfh cells have been shown to be the limiting cell subset in the GC reaction and critical for the B cell maturation processes and production of high affinity antibodies [51][52][53].Our study specifically aimed to investigate whether altering the magnitude of memory CD4+ T cell help in the GC reaction would enhance the generation of antiviral humoral immunity to primary influenza infection.Previous studies have established that lineage-committed memory Th1 and Tfh cells generated during intracellular pathogenic infections can be specifically recalled upon subsequent challenges [67,69,[86][87][88].In addition, increases in Tfh cells have been shown to positively correlate with increases in GC B cell magnitude and broadly neutralizing antibodies in response to viral infections and vaccinations [43][44][45][46][47][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103][104][105].Preclinical studies investigating novel vaccination strategies successfully targeted increases in antigen-specific Tfh cells and GC and humoral responses [106][107][108][109], signifying the importance of targeting CD4+ T cells in the GC and production of high affinity antibodies.However, vaccination strategies or adjuvants specifically to target the recall of CD4+ Tfh cells to enhance the GC and its products have been slow to develop beyond the preclinical stage.We found that specifically targeting the recall and expansion of memory antigen-specific CD4+ T cells induced an increase in GC Tfh and HA-specific GC B cells early compared to mice that lacked antigen-specific memory CD4+ T cell during primary influenza infection, indicating that our findings concur with previous studies that targeting CD4+ T cells is a successful strategy to enhance the GC reaction [43,47,[110][111][112].Similarly, MacLeod et al. (2011) found that heterologously-primed antigenspecific memory CD4+ T cells enhanced early primary GC B cell and antibody responses compared to naïve CD4+ T cells, even when similar numbers of naïve CD4+ T cells were adoptively transferred into mice [111].In a chronic LCMV infection model, He et al. (2018) similarly used a CD4+ T cell epitope-based heterologous prime-boost strategy and showed an increase in antigen-specific Tfh cells, total GC B cells, and LCMV-specific antibody titers [112].In our study, while heterologous priming enhanced the early GC Tfh and GC B cell magnitude, we did not see increases in HA-specific antibody titers, but we did see selection for specific clones in the resultant B cell repertoires.Overall, our study demonstrates that increasing the amount of antigen-specific Tfh cell help can drive an increase in the size of the germinal center response to infection, indicating that future studies could use heterologous infection or immunization priming of T helper cells to improve humoral immune responses.
Previous studies investigating T cell responses after intranasal immunization showed induction of protective proinflammatory lung-resident antigen-specific CD4+ and CD8+ T cells early after influenza challenge [73,[113][114][115][116][117].As virus-and vaccine-induced lung-resident CD4+ T RM cells have been shown to mediate protection from influenza infection [70][71][72][73][74], it is important to understand how heterologous infection or immunization priming of CD4+ T RM cells and resultant subsets of T RM cells could enhance localized immune responses.CD4 + T RM , specifically resident Tfh cells, have also been shown to be important in the formation of and promotion of CD8+ T cell and B cell localization to inducible BALT structures [75,[118][119][120].Regarding the importance of CD4+ T cells in formation of iBALT tertiary germinal center-like structures [121][122][123][124][125][126], and that we saw increases in lung-resident Th1 and Tfh-like cells in heterologously primed mice, the differences in cellular composition or iBALT formation kinetics with either LCMV infection or adjuvanted rGP immunization priming compared to primary influenza infection warrants further investigation.As we have previously shown, non-Tfh cell populations are different between adjuvanted rGP immunization and LCMV infection [63], investigating the CD4+ T RM subsets resultant from these priming strategies and their distinct roles in their recall during an influenza challenge poses an interesting question, as IFNγsecreting CD4+ T cells have been shown to be protective against influenza infection in secondary recalled responses [127,128].
Prior studies investigating the recall of memory CD8+ T cells in heterosubtypic influenza infection have shown that protective CD8+ T RM cells were found to undergo robust clonal expansion after secondary infection and express large amounts IFNγ, though the secondary effectors were dominated by recognition of a single immunodominant epitope [129][130][131][132][133]. As one study found neither infection of the lung nor antigen persistence was required for establishment in the lung of antigen-specific CD8+ T cells [131], we found similar results in our study investigating CD4+ T cells as adjuvanted rGP immunization showed minimal lung-resident memory CD4+ T cells prior to influenza challenge but had significantly expanded secondary effector CD4+ T cells and CD4+ T RM in the lung compared to primary influenza infection, suggesting either increased trafficking to the lung or a larger antigen-specific memory T RM pool compared to naïve mice.Antigen-specific CD8+ T cells that are cross-reactive between heterosubtypic influenza infections can restrict the influenza challenge, resulting in the lack of a boosted CD4 T cell response to the secondary challenge (130,131).Therefore, our studies provide unique insight into secondary CD4+ T cell recall responses that are not blunted by pre-existing CD8+ T cell recall responses.
In agreement with the idea that Tfh cells are the limiting cell subset in the GC reaction and the generation of GC-derived products, following heterologous influenza rechallenge of memory CD4+ T cells we saw an early increased magnitude of antigen-specific GC Tfh and GC B cells.However, additional studies are needed to assess the direct impact of heterologous infection or immunization priming of CD4+ T cells on survival, protection, or enhancing production of cross-reactive high affinity antibodies in response to influenza challenge.By investigating GC Tfh cell involvement in the enhancement of antiviral humoral immune responses, it is evident that new vaccination strategies should be specifically designed to engage memory CD4+ T cells to enhance the GC.Furthermore, our findings that heterologous infection or immunization priming increased expansion of localized lung antigen-specific CD4 + immune responses and lung T RM populations suggest understanding differences in the lungresident CD4+ T cell responses induced by vaccination versus previous viral infection may also be important in novel vaccine design.Ultimately, future studies are necessary to determine the mechanisms into the direct involvement of naïve versus pre-existing memory Tfh cells in preferentially generating universal and broadly neutralizing antibodies to enhance protection against influenza infection or in development of novel vaccine strategies. .B cell receptor sequences.Tab 1 indicates the amino acid sequence, nucleotide sequence, frequency and number of productive rearrangements, and annotation for V, D, and J regions.Tab 2 indicates the frequency of individual B cell receptors in each sample.(XLSX)

S1 Table
Figures were created with the Immunarch package or GraphPad Prism version 9.4.1 for macOS.The full sequencing dataset is available in S1 Table.

Fig 6 .Fig 7 .
Fig 6.Generation of memory CD4+ T cells by heterologous immunization enhanced long-term memory Th1 and CD4+ T RM cells following influenza infection.Flow cytometry analysis of CD45-i.v.negative CD4+ T cells from lung 42 days after PR8-HA-GP 61-80 influenza virus infection in heterologously primed mice (GP(1˚)PR8(2˚), filled triangle, or LCMV(1˚)PR8(2˚), filled diamond), homologously primed mice (PR8(1˚)PR8(2˚), filled hexagon) or unprimed naïve mice (PR8(1˚), unfilled circle).CD4+ T cells were analyzed by staining with I-A b :gp66-77 tetramer.(A) Representative flow plots of CD4 and tetramer analysis of total CD4+ T cells.(B) Number of tetramer+CD44+ of total CD4+ T cells.(C) Representative flow plots of CD69 analysis gated on tetramer+CD4+ T cells.(D) Number of CD69+ T RM cells of total tetramer+CD4+ T cells.(E) Representative flow plots of FR4 and LY6C analysis gated on tetramer+CD4+ T cells.(F) Number of LY6C +FR4-(Th1) cells of total tetramer+CD4+ T cells.(G) Ratio of the number of tetramer+ LY6C+FR4-(Th1) cells to number of tetramer+ LY6C-FR4+ (Tfh) cells.n � 3 per group per experiment at each timepoint.Data shown are from one experiment and are representative of two to three independent experiments.Statistically significant p values of <0.05 are indicated and were determined using Mann-Whitney U test.Error bars represent Mean±SEM, *p�0.05,**p�0.01,***p�0.001,****p�0.0001.https://doi.org/10.1371/journal.ppat.1011639.g006 package (Fig 7F).We found trending differences in clonotype proportions, including increased proportions of the CARGGYW and CARGTYW clones and a lack of the CARGGYDGYYGAMDYW clone in the GP(1˚)PR8(2˚) group (Fig 7F).In addition, the CARHEVSYWYFDVW clone was found in mice only infected with PR8-HA-GP 61-80 (3 of 5 PR8(1˚) mice and 4 of 5 PR8(1˚)PR8(2˚) mice) (Fig 7F).Only the CARGAYW clone was shared in all 20 infected mice, and thus had the largest representation by proportion (Fig 7F).Additionally, this clone was not contained in our control naïve CD19+Fas-IgD+ B cells.We then analyzed the 10 clonotypes largest by proportion in each priming strategy group shared in at least 2 of 5 mice (Fig 7G-7J).Our data showed that the largest shared clone, CARGAYW, was less represented proportionally in the PR8(1˚) group while unique clones, including CVQMEERPPLFTYW, were more largely represented (Fig 7G).In addition, our data show the 10 proportionally largest clones in the PR8(1˚) group comprised over 2-fold more of the total pooled repertoire proportion (>4% total) compared to the other three groups (all <2% total) (Fig 7G), and with the Morisita overlap data (Fig 7E) suggests a more unique repertoire for the PR8(1˚) group.Together, these data show that while the plasmablast repertoires of individual mice were dominated by unique clones, analyses of the partially shared clones among individual mice were able to characterize differences in the representation of specific clonal sequences by priming strategy.